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Cobos R.,University of Leon | Barreiro C.,Institute Biotecnologia Of Leon Inbiotec | Mateos R.M.,University of Leon | Coque J.R.,University of Leon
Proteome Science

Background: The phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight) and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline). This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome.Results: Intracellular and secreted proteins from D. seriata collected from liquid cultures were separated using two-dimensional gel electrophoresis. About 550 cytoplasmic proteins were reproducibly present in 3 independent extractions, being 53 identified by peptide mass fingerprinting and tandem mass spectrometry. The secretome analysis showed 75 secreted proteins reproducibly present in 3 biological replicates, being 16 identified. Several of the proteins had been previously identified as virulence factors in other fungal strains, although their contribution to pathogenicity in D. seriata remained to be analyzed. When D. seriata was grown in a medium supplemented with carboxymethylcellulose, 3 proteins were up-regulated and 30 down-regulated. Within the up-regulated proteins, two were identified as alcohol dehydrogenase and mitochondrial peroxyrredoxin-1, suggesting that they could play a significant role in the pathogenicity process. As for the 30 down-regulated proteins, 9 were identified being several of them involved in carbohydrate metabolism.Conclusions: This study is the first report on proteomics on D. seriata. The proteomic data obtained will be important to understand the pathogenicity process. In fact, several of the identified proteins have been reported as pathogenicity factors in other phytopathogenic fungi. Moreover, this proteomic analysis supposes a useful basis for deepening into D. seriata knowledge and will contribute to the development of the molecular biology of this fungal strain as it has been demonstrated by cloning the gene Prx1 encoding mitochondrial peroxiredoxin-1 of D. seriata (the first gene to be cloned in this microorganism; data not shown). © 2010 Cobos et al; licensee BioMed Central Ltd. Source

Barreiro C.,Institute Biotecnologia Of Leon Inbiotec | Martinez-Castro M.,Gadea Biopharma SL Parque Tecnologico de Leon
Applied Microbiology and Biotechnology

The current off-patent state of tacrolimus (FK506) has opened the hunting season for new generic pharmaceutical formulations of this immunosuppressant. This fact has boosted the scientific and industrial research on tacrolimus for the last 5 years in order to improve its production. The fast discovery of tacrolimus producer strains has generated a huge number of producers, which presents the biosynthetic cluster of FK506 as a high promiscuous genetic region. For the first time, the current state-of-the-art on the tacrolimus biosynthesis, production improvements and drug purification is reviewed. On one hand, all the genes involved in the tacrolimus biosynthesis, in addition to the traditional PKS/NRPS, as well as their regulation are analysed. On the other hand, tacrolimus direct and indirect precursors are reviewed as a straight manner to improve the final yield, which is a current trend in the field. Twenty years of industrial and scientific improvements on tacrolimus production are summarised, whereas future trends are also drafted. © 2013 Springer-Verlag Berlin Heidelberg. Source

Recio E.,Institute Biotecnologia Of Leon Inbiotec | Alvarez-Rodriguez M.L.,University of Leon | Rumbero A.,Autonomous University of Madrid | Garzon E.,University of Leon | Coque J.J.R.,University of Leon
Journal of Agricultural and Food Chemistry

A chemical method for the efficient destruction of 2,4,6-trichloroanisole (TCA) and pentachloroanisole (PCA) in aqueous solutions by using hydrogen peroxide as an oxidant catalyzed by molybdate ions in alkaline conditions was developed. Under optimal conditions, more than 80.0% TCA and 75.8% PCA were degraded within the first 60 min of reaction. Chloroanisoles destruction was followed by a concomitant release of up to 2.9 chloride ions per TCA molecule and 4.6 chloride ions per PCA molecule, indicating an almost complete dehalogenation of chloroanisoles. This method was modified to be adapted to chloroanisoles removal from the surface of cork materials including natural cork stoppers (86.0% decrease in releasable TCA content), agglomerated corks (78.2%), and granulated cork (51.3%). This method has proved to be efficient and inexpensive with practical application in the cork industry to lower TCA levels in cork materials. © 2011 American Chemical Society. Source

O'Callaghan J.,University College Cork | Coghlan A.,University College Cork | Abbas A.,University College Cork | Garcia-Estrada C.,Institute Biotecnologia Of Leon Inbiotec | And 3 more authors.
International Journal of Food Microbiology

The ochratoxin A (OTA) polyketide synthase otapks gene has been cloned from Penicillium verrucosum. A P. verrucosum mutant in which the otapksPV gene has been interrupted cannot synthesize ochratoxin A. The protein is most similar to the citrinin polyketide synthase CtnpksMa from Monascus anka (83% identity at the amino acid level). Different nutritional conditions influence OTA production in P. verrucosum, with the addition of glycerol and galactose to MCB resulting in approximately 19 and 32 fold increases in OTA production respectively. These effects are mirrored in increased levels of otapksPV gene transcription. In contrast, the addition of glucose to MCB containing galactose results in an approximate 10 fold repression in OTA production, with this repression again being mirrored in decreased levels of otapksPV gene transcription. Thus the effects of different carbon sources on OTA production in P. verucosum appear to be regulated at the level of gene transcription. Two additional open reading frames, otaE and otaT, were identified in the 5' and 3' flanking regions of otapksPV, respectively. The otaT and otaE genes are co-expressed with P. verrucosum otapksPv, indicating a possible role for these genes in OTA biosynthesis. Furthermore, otaT and otaE were identified as putative homologues of the M. anka citrinin transporter ctnC (72% amino acid identity) and M. anka citrinin oxidoreductase ctnB (83% amino acid identity); suggesting that the genes involved in OTA production in P. verrucosum may be very similar to those involved in citrinin production in M. anka. © 2012 Elsevier B.V. Source

Garcia-Estrada C.,Institute Biotecnologia Of Leon Inbiotec | Ullan R.V.,Institute Biotecnologia Of Leon Inbiotec | Albillos S.M.,Institute Biotecnologia Of Leon Inbiotec | Fernandez-Bodega M.A.,Institute Biotecnologia Of Leon Inbiotec | And 5 more authors.
Chemistry and Biology

A single gene cluster of Penicillium chrysogenum contains genes involved in the biosynthesis and secretion of the mycotoxins roquefortine C and meleagrin. Five of these genes have been silenced by RNAi. Pc21g15480 (rds) encodes a nonribosomal cyclodipeptide synthetase for the biosynthesis of both roquefortine C and meleagrin. Pc21g15430 (rpt) encodes a prenyltransferase also required for the biosynthesis of both mycotoxins. Silencing of Pc21g15460 or Pc21g15470 led to a decrease in roquefortine C and meleagrin, whereas silencing of the methyltransferase gene (Pc21g15440; gmt) resulted in accumulation of glandicolin B, indicating that this enzyme catalyzes the conversion of glandicolin B to meleagrin. All these genes are transcriptionally coregulated. Our results prove that roquefortine C and meleagrin derive from a single pathway. © 2011 Elsevier Ltd All rights reserved. Source

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